Sphingosine pathway modulating compounds for the treatment of cancers

ABSTRACT

The invention provides methods and compositions for treating cancers and myeloproliferative disorders using sphingosine kinase-1 inhibitors, such as SK1-I, and selective sphingosine-1-phosphate receptor agonists, such as ozanimod.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/842,814 filed Apr. 8, 2020, which is a divisional of U.S. applicationSer. No. 16/229,490, filed Dec. 21, 2018 (now U.S. Pat. No. 10,669,879),which is a continuation-in-part of U.S. application Ser. No. 16/017,303filed Jun. 25, 2018 (now U.S. Pat. No. 10,675,255), which claims thebenefit of U.S. provisional patent application Ser. No. 62/524,221 filedJun. 23, 2017, each of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to the field of pharmaceutical treatment ofcancers.

BACKGROUND

Sphingosine-1-phosphate (S1P) was discovered to be a bioactive signalingmolecule over 20 years ago. Studies have since identified two relatedkinases, sphingosine kinase 1 and 2 (a/k/a sphingosine kinase “type I”and “type II” respectively, and SphK1 and SphK2 respectively), whichcatalyze the phosphorylation of sphingosine to S1P. Extracellular S1Pcan bind to and activate each of five S1P-specific, G protein-coupledreceptors (designated S1PR₁₋₅) to regulate cellular and physiologicalprocesses in an autocrine or paracrine manner. Selective inhibitors ofeach of sphingosine kinase 1 and 2, as well as both non-selective andselective agonists of S1PRs, have been developed and are known in theart.

SUMMARY

One embodiment of the invention provides a method for treating livercancer, such as hepatocellular carcinoma (HCC), in a mammalian subject,such as a human, that includes the step of:

-   -   administering to a mammalian subject in need of treatment for        liver cancer, a therapeutically effective amount of a        sphingosine kinase type I inhibitor, such as SK1-I or a        pharmaceutically acceptable salt thereof.

A related embodiment of the invention provides a pharmaceuticalcomposition that includes a sphingosine kinase type I inhibitor, such asSK1-I or a pharmaceutically acceptable salt thereof for the treatment ofliver cancer, such as HCC, in a mammal, such as a human patient.

Another embodiment of the invention provides a method for treating acancer or a myeloproliferative disorder (myeloproliferative neoplasm) ina mammalian subject, such as a human, that includes the step of:

-   -   administering to a mammalian subject in need of treatment for a        cancer or myeloproliferative disorder, a therapeutically        effective amount of a sphingosine-1-phosphate receptor agonist,        such as an agonist of one or both of sphingosine-1-phosphate        receptor-1 (S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅)        such as ozanimod (RPC1063) or a pharmaceutically acceptable salt        thereof, or an active metabolite of ozanimod or a        pharmaceutically acceptable salt thereof.

A related embodiment of the invention provides a pharmaceuticalcomposition for the treatment of a cancer or a myeloproliferativedisorder (myeloproliferative neoplasm) in a mammalian subject, such as ahuman, that includes:

-   -   a therapeutically effective amount of a sphingosine-1-phosphate        receptor agonist, such as an agonist of one or both of        sphingosine-1-phosphate receptor-1 (S1P₁) and        sphingosine-1-phosphate receptor-5 (S1P₅) such as ozanimod        (RPC1063) or a pharmaceutically acceptable salt thereof, or an        active metabolite of ozanimod or a pharmaceutically acceptable        salt thereof.

Still another embodiment of the invention provides a method for treatinga cancer or a myleoproliferative disorder (myeloproliferative neoplasm),such as any of those disclosed herein, in a mammalian subject, such as ahuman, including the step of:

-   -   co-administering to a mammalian subject in need of treatment for        a cancer or myeloproliferative disorder, a therapeutically        effective amount of:        -   (a) a sphingosine kinase type I inhibitor, such as one            disclosed in U.S. Pat. Nos. 8,372,888 and/or 8,314,151, such            as SK1-I, or a pharmaceutically acceptable salt thereof; and        -   (b) one or more immune checkpoint inhibitors, which may be            monoclonal antibodies, such as one or more selected from the            group consisting of: PD-1 inhibitors such as mAbs            Pembrolizumab (Keytruda®) and Nivolumab (Opdivo®); PD-L1            inhibitors such as mAbs Atezolizumab (Tecentriq®), Avelumab            (Bavencio®), and Durvalumab (Imfinzi®); and CTLA-4            inhibitors such as mAb Ipilimumab (Yervoy®); and V-domain Ig            Suppressor of T Cell Activation (VISTA) inhibitors such as            mAb JNJ-61610588 (ImmuNext Inc.).

Additional features, advantages, and embodiments of the invention may beset forth or apparent from consideration of the following detaileddescription, drawings if any, and claims. Moreover, it is to beunderstood that both the foregoing summary of the invention and thefollowing detailed description are exemplary and intended to providefurther explanation without limiting the scope of the invention asclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows MTT assay data (72 hours) for various concentrations ofozanimod for four human hepatocellular carcinoma cell lines.

FIG. 2 shows MTT assay data (72 hours) for various concentrations ofABC294640 for four human hepatocellular carcinoma cell lines.

FIG. 3 shows MTT assay data (72 hours) for various concentrations ofSK1-I for four human hepatocellular carcinoma cell lines.

FIG. 4A shows MTT assay data (48 hours) for various concentrations ofSK1-I for three human hepatocellular carcinoma cell lines. FIG. 4B showsMTT assay data (48 hours) for various concentrations of PF-543, a superpotent SphK1 inhibitor, for the same three human hepatocellularcarcinoma cell lines shown in FIG. 4A.

FIGS. 5A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for Huh7 cells. SK1-I strongly induced apoptosis inthe Huh7 cells.

FIGS. 6A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for PLC-PRF5 cells. SK1-I strongly induced apoptosisin the PLC-PRF5 cells.

FIGS. 7A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for Hep 3B cells. SK1-I strongly induced apoptosisin the Hep 3B cells.

FIG. 8A-D show apoptosis assay data for various concentrations of SK1-Iand no-drug control for Hep G2 cells. SK1-I strongly induced apoptosisin the Hep G2 cells.

FIGS. 9A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for Huh7 cells. Ozanimod strongly inducedapoptosis in the Huh7 cells.

FIGS. 10A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for PLC-PRF5 cells. Ozanimod stronglyinduced apoptosis in the PLC-PRF5 cells.

FIGS. 11A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep 3B cells. Ozanimod strongly inducedapoptosis in the Hep 3B cells.

FIGS. 12A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep G2 cells. Ozanimod strongly inducedapoptosis in the Hep G2 cells.

FIGS. 13A-D shows apoptosis assay data for various concentrations ofABC294640 and no-drug control for Huh7 cells. ABC294640 failed to induceapoptosis in the Huh7 cells.

FIGS. 14A-D show apoptosis assay data for various concentrations ofABC294640 and no-drug control for PLC-PRF5 cells. ABC294640 did notsubstantially induce apoptosis in the PLC-PRF5 cells.

FIGS. 15A-D show apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep 3B cells. ABC294640 did notsubstantially induce apoptosis in the Hep 3B cells.

FIGS. 16A-D show apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep G2 cells. ABC294640 failed toinduce apoptosis in the Hep G2 cells.

FIGS. 17A-C show apoptosis assay data for various concentrations ofSK1-I and no-drug control for Jurkat cells (human T-cell leukemia cellline). SK1-I strongly induced apoptosis in the Jurkat cells.

FIGS. 18A-C show apoptosis assay data for various concentrations ofozanimod and no-drug control for Jurkat cells. Ozanimod strongly inducedapoptosis in the Jurkat cells.

FIGS. 19A-D show apoptosis assay data for various concentrations ofSK1-I and no-drug control for primary human hepatocytes. SK1-I did notinduce apoptosis of the primary human hepatocytes at any concentrationtested.

FIGS. 20A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for primary human hepatocytes. Ozanimod didnot induce apoptosis of the primary human hepatocytes at anyconcentration tested.

FIG. 21 shows the cell viability effects of 5 μM SK1-I alone, 5 μMozanimod alone, and the combination of 5 μM SK1-I and 5 μM ozanimod onJurkat cells, Jurkat cells cultured with IL-2, PBMCs, and PBMCs culturedwith IL-2.

FIG. 22A shows the cell viability effects of different concentrations ofSK1-I and ozanimod, each alone and in combination, on pancreatic cellline PanC-1 cells. FIG. 22B shows the cell viability effects ofdifferent concentrations of SK1-I and ozanimod, each alone and incombination, on pancreatic cancer cell line BxPC-3 cells.

DETAILED DESCRIPTION

The invention provides new uses of sphingosine kinase-1 inhibitors, suchas SK1-I, and selective sphingosine-1-phosphate receptor agonists, suchas ozanimod, for treating cancers, such as a liver cancer, andmyeloproliferative neoplasms (myeloproliferative disorders), in mammals,such as human patients. The invention also provides new uses ofselective sphingosine kinase type I inhibitors, such as SK1-I, andselective sphingosine-1-phosphate receptor agonists, such as ozanimod,for inducing apoptosis and/or necrosis of mammalian, such as human,cancer cells, such as liver cancer cells, and myeloproliferativeneoplasm cells.

Sphingosine kinase 1 inhibitors used in various embodiments of theinvention may, for example, include any of those disclosed in U.S. Pat.Nos. 8,372,888 and/or 8,314,151, each of which is hereby incorporated byreference in its entirety herein, or pharmaceutically acceptable saltsthereof. The sphingosine kinase I inhibitor may, for example, be(E,2R,3S)-2-(methylamino)-5-(4-pentylphenyl)pent-4-ene-1,3-diol (alsoknown as SK1-I), or a pharmaceutically acceptable salt thereof such asbut not limited to a hydrochloride salt. The structure of SK1-I is shownbelow.

See also Paugh et al., Blood, 2008 112: 1382-1391.

The sphingosine kinase I inhibitor may, for example, be a compoundhaving the structure

-   -   or a pharmaceutically acceptable salt of the compound such as        but not limited to a hydrochloride salt.

The sphingosine kinase I inhibitor may, for example, be a compoundhaving the structure

-   -   wherein R is selected from a straight carbon chain, a branched        carbon chain, a straight carbon chain comprising one or more        heteroatoms, a branched carbon chain comprising one or more        heteroatoms, a cyclic ring, a heterocyclic ring, an aromatic        ring, a hetero-aromatic ring, or any combination of the        foregoing,    -   or a pharmaceutically acceptable salt thereof such as but not        limited to a hydrochloride salt.

The sphingosine kinase I inhibitor may, for example, be a compoundhaving the structure

-   -   wherein R is 3,4-dimethoxy, 4-phenyl or 3-pentyl,    -   or a pharmaceutically acceptable salt thereof such as but not        limited to a hydrochloride salt.

Sphingosine-1-phosphate receptor agonists used in various embodiments ofthe invention may, for example, be any of those disclosed in any of U.S.Pub. Nos. 20110172202, 20130231326, and 20150299149, or pharmaceuticallyacceptable salts thereof. The agonists may be agonists of one or both ofsphingosine-1-phosphate receptor-1 (S1P₁) and sphingosine-1-phosphatereceptor-5 (S1P₅) and may have little or at least no substantial agonistactivity against other sphingosine-1-phosphate receptors (in a mammalsuch as a human). The sphingosine-1-phosphate receptor agonist used may,for example, be5-[3-[(1S)-1-(2-hydroxyethylamino)-2,3-dihydro-1H-inden-4-yl]-1,2,4-oxadiazol-5-yl]-2-propan-2-yloxybenzonitrile(also known as ozanimod and RPC1063) or a pharmaceutically acceptablesalt thereof such as but not limited to a hydrochloride salt. Thestructure of ozanimod is shown below.

See also Scott et al., British Journal of Pharmacology 2016173:1778-1792.

The sphingosine-1-phosphate receptor agonist may, for example, beetrasimod or a pharmaceutically acceptable salt thereof such as but notlimited to a hydrochloride salt. The structure of etrasimod is shownbelow.

The sphingosine-1-phosphate receptor agonist may, for example, beamiselimod or a pharmaceutically acceptable salt thereof such as but notlimited to a hydrochloride salt. The structure of amiselimod is shownbelow.

ABC294640 (also known as Yeliva®) used in the experiments presentedherein is a reported sphingosine kinase-2 selective inhibitor, namelythe compound (7S)-3-(4-chlorophenyl)-N-(pyridin-4-ylmethyl)adamantane-1-carboxamide.The structure of ABC294640 is shown below.

See also French et al., J. Pharmacol. Exp. Ther. 2010, 333, 129-139.

EXPERIMENTS

MTT cell viability assays evaluating the effect of differentconcentrations of each of ozanimod, ABC294640 and SK1-I on four humanhepatocellular carcinoma cell lines, Hep G2, Hep 3B, Huh 7 and PLC-PRF5were performed. These four cells line were selected for the studybecause they are among HCC cells lines whose gene expression profilesmost closely resemble those of primary HCC tumors. See Chen et al., BMCMedical Genomics 2015, 8 (Suppl 2):S5. The following concentrations ofthe compounds were tested.

-   -   Ozanimod: 200 μM, 111.1 μM, 61.73 μM, 34.29 μM, 19.05 μM, 10.58        μM, and 0 μM.    -   ABC294640: 200 μM, 111.1 μM, 61.73 μM, 34.29 μM, 19.05 μM, 10.58        μM, and 0 μM.    -   SK1-I: 20 μM, 11.11 μM, 6.173 μM, 3.429 μM, 1.905 μM, 1.058 μM,        and 0 μM.

The following MTT assay protocol was followed.

-   -   Prepared stock solutions: 50 mM ozanimod, 50 mM ABC294640, 10 mM        SK1-I in DMSO.    -   Plated 20000 cells in 160 μl medium per well in 96-well plates        for each cell line and incubated at 37° C. overnight.    -   Prepared compound serial dilutions: for ozanimod and ABC294640:        dilute stock 50 mM 1:50 in medium to 1000 μM; for SK1-I, dilute        stock 10 mM 1:50 in medium to 100 them make serial 1:1.8 fold        serial dilution to the titration. For negative control, diluted        DMSO 1:50 to medium.    -   Added 40 μl negative control and serially titrated compounds to        160 μl of cells for each cell line. Performed in triplicate for        each cell line.    -   Incubated at 37° C. for 72 hours.    -   Performed assay using Vybrant MTT Cell Proliferation Assay kit        (V-13154) (Molecular Probes) from Thermo Fisher Scientific        (Waltham, MA USA).        The results of these 72-hour treatment MTT assays are shown in        FIGS. 1-3 as follows.

FIG. 1 shows the MTT assay data (72 hours) for ozanimod for the fourhuman hepatocellular carcinoma cell lines.

FIG. 2 shows the MTT assay data (72 hours) for ABC294640 for the fourhuman hepatocellular carcinoma cell lines.

FIG. 3 shows the MTT assay data (72 hours) for SK1-I for the four humanhepatocellular carcinoma cell lines.

48-hour treatment MTT assays were also performed as follows. FIG. 4Ashows MTT assay data (48 hours) for various concentrations of SK1-I forthree human hepatocellular carcinoma cell lines, Hep G2, Huh 7, andPLC-PRF5. FIG. 4B shows MTT assay data (48 hours) for variousconcentrations of PF-543, a super potent SphK1 inhibitor (see Schnute etal., Biochem. J. (2012) 444, 79-88), for the same three humanhepatocellular carcinoma cell lines shown in FIG. 4A. This data showsthat SK1-I is more effective at killing hepatocellular carcinoma cellsthan PF-543 despite the latter drug's much greater potency in inhibitingSphK1.

Apoptosis/necrosis assays evaluating the effect of differentconcentrations of each of ozanimod, ABC294640 and SK1-I on the fourhuman hepatocellular carcinoma cell lines, Hep G2, Hep 3B, Huh 7 andPLC-PRF5 were also performed.

The protocol used for the apoptosis assays was:

-   -   Plated cells in 6-well plates and incubated at 37° C. overnight.    -   Prepared concentrations of test compound and control compound        DMSO in media.        -   Ozanimod: 5 μM, 10 μM, and 20 μM.        -   ABC294640: 10 μM, 20 μM, and 40 μM.        -   SK1-I: 10 μM, 20 μM, and 40 μM.        -   Control: 0.2% DMSO.    -   Aspirated the medium from the 6-well plates and added test        compound concentrations in media or 0.2% DMSO control in media.        Incubated at 37° C. for 24 hours.    -   Collected and processed the cells following the flow cytometry        protocol of the GFP Certified® Apoptosis/Necrosis detection kit        from Enzo Life Sciences, Inc. (product no. ENZ-51002;        Farmingdale, NY, USA).

FIGS. 5-16 present graphs plotting the data from these 24-hour apoptosisassays for the different concentrations of compounds and no-drug controlfor the various cell lines. Channel FL2 picks up the apoptosis signaland channel FL3 picks up the necrosis signal. Data points in Quadrant 3(Q3) in the graphs corresponds to cells undergoing apoptosis (cellspositive for the apoptosis detection reagent of the assay). Data pointsin Quadrant 2 (Q2) in the graphs corresponds to cells that are positivefor the apoptosis detection reagent and positive for the necrosisdetection reagent of the assay (indicative of late-stage apoptosis).

FIGS. 5A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for Huh7 cells. FIG. 5A shows the results forno-drug control. FIG. 5B shows the results for treatment with 10 μMSK1-I. FIG. 5C shows the results for treatment with 20 μM SK1-I. FIG. 5Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the Huh7 cells.

FIGS. 6A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for PLC-PRF5 cells. FIG. 6A shows the resultsfor no-drug control. FIG. 6B shows the results for treatment with 10 μMSK1-I. FIG. 6C shows the results for treatment with 20 μM SK1-I. FIG. 6Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the PLC-PRF5 cells.

FIGS. 7A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for Hep 3B cells. FIG. 7A shows the resultsfor no-drug control. FIG. 7B shows the results for treatment with 10 μMSK1-I. FIG. 7C shows the results for treatment with 20 μM SK1-I. FIG. 7Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the Hep 3B cells.

FIGS. 8A-D show the apoptosis assay data for various concentrations ofSK1-I and no-drug control for Hep G2 cells. FIG. 8A shows the resultsfor no-drug control. FIG. 8B shows the results for treatment with 10 μMSK1-I. FIG. 8C shows the results for treatment with 20 μM SK1-I. FIG. 8Dshows the results for treatment with 40 μM SK1-I. SK1-I strongly inducedapoptosis in the Hep G2 cells.

FIGS. 9A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for Huh7 cells. FIG. 9A shows the resultsfor no-drug control. FIG. 9B shows the results for treatment with 5 μMozanimod. FIG. 9C shows the results for treatment with 10 μM ozanimod.FIG. 9D shows the results for treatment with 20 μM ozanimod. Ozanimodstrongly induced apoptosis in the Huh7 cells.

FIGS. 10A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for PLC-PRF5 cells. FIG. 10A shows theresults for no-drug control. FIG. 10B shows the results for treatmentwith 5 μM ozanimod. FIG. 10C shows the results for treatment with 10 μMozanimod. FIG. 10D shows the results for treatment with 20 μM ozanimod.Ozanimod strongly induced apoptosis in the PLC-PRF5 cells.

FIGS. 11A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep 3B cells. FIG. 11A shows theresults for no-drug control. FIG. 11B shows the results for treatmentwith 10 μM ozanimod. FIG. 11C shows the results for treatment with 20 μMozanimod. FIG. 11D shows the results for treatment with 40 μM ozanimod.Ozanimod strongly induced apoptosis in the Hep 3B cells.

FIGS. 12A-D show the apoptosis assay data for various concentrations ofozanimod and no-drug control for Hep G2 cells. FIG. 12A shows theresults for no-drug control. FIG. 12B shows the results for treatmentwith 10 μM ozanimod. FIG. 12C shows the results for treatment with 20 μMozanimod. FIG. 12D shows the results for treatment with 40 μM ozanimod.Ozanimod strongly induced apoptosis in the Hep G2 cells.

FIGS. 13A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for Huh7 cells. FIG. 13A shows the resultsfor no-drug control. FIG. 13B shows the results for treatment with 10 μMABC294640. FIG. 13C shows the results for treatment with 20 μMABC294640. FIG. 13D shows the results for treatment with 40 μMABC294640. ABC294640 failed to induce apoptosis in the Huh7 cells.

FIGS. 14A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for PLC-PRF5 cells. FIG. 14A shows theresults for no-drug control. FIG. 14B shows the results for treatmentwith 10 μM ABC294640. FIG. 14C shows the results for treatment with 20μM ABC294640. FIG. 14D shows the results for treatment with 40 μMABC294640. ABC294640 did not substantially induce apoptosis in thePLC-PRF5 cells.

FIGS. 15A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep 3B cells. FIG. 15A shows theresults for no-drug control. FIG. 15B shows the results for treatmentwith 10 μM ABC294640. FIG. 15C shows the results for treatment with 20μM ABC294640. FIG. 15D shows the results for treatment with 40 μMABC294640. ABC294640 did not substantially induce apoptosis in the Hep3B cells.

FIGS. 16A-D show the apoptosis assay data for various concentrations ofABC294640 and no-drug control for Hep G2 cells. FIG. 16A shows theresults for no-drug control. FIG. 16B shows the results for treatmentwith 10 μM ABC294640. FIG. 16C shows the results for treatment with 20μM ABC294640. FIG. 16D shows the results for treatment with 40 μMABC294640. ABC294640 failed to induce apoptosis in the Hep G2 cells.

FIGS. 17A-C show apoptosis assay data for various concentrations ofSK1-I and no-drug control for Jurkat cells (human T-cell leukemia cellline). FIG. 17A shows the results for no-drug control. FIG. 17B showsthe results for treatment with 10 μM SK1-I. FIG. 17C shows the resultsfor treatment with 20 μM SK1-I. SK1-I strongly induced apoptosis in theJurkat cells.

FIGS. 18A-C show apoptosis assay data for various concentrations ofozanimod and no-drug control for Jurkat cells. FIG. 18A shows theresults for no-drug control. FIG. 18B shows the results for treatmentwith 5 μM ozanimod. FIG. 18C shows the results for treatment with 10 μMozanimod. Ozanimod strongly induced apoptosis in the Jurkat cells.

In still further experiments, the effects of SK1-I and ozanimod onnormal (non-cancerous) primary human hepatocytes were investigated.Fresh human hepatocytes in a 12-well plate (HUF12) were obtained fromTriangle Research Labs (Durham, NC, USA; part of Lonza Group) andhandled according to the supplier's protocol. The shipping medium wasaspirated from each well and replaced with 1 ml per well of warmHepatocyte Maintenance Medium. The plate was then placed in a 5% CO₂incubator at 37° C. and the hepatocytes were allowed to acclimateovernight. The Hepatocyte Maintenance Medium was replaced beforetreatment with drug or no-drug control, and the hepatocytes were treatedwith 0 μM (no-drug control), 10 μM, 20 μM, or 40 μM SK1-I or 0 μM, 2.5μM, 5 μM, or 10 μM ozanimod for 24 hours under incubation. The cellswere then harvested and analyzed using the GFP Certified®Apoptosis/Necrosis detection kit. The results are shown in FIGS. 19A-Dfor SK1-I and FIGS. 20A-D for ozanimod as follows.

FIGS. 19A-D show apoptosis assay data for various concentrations ofSK1-I and no-drug control for primary human hepatocytes. FIG. 19A showsthe results for no-drug control. FIG. 19B shows the results fortreatment with 10 μM SK1-I. FIG. 19C shows the results for treatmentwith 20 μM SK1-I. FIG. 19D shows the results for treatment with 40 μMSK1-I. SK1-I did not induce apoptosis of the primary human hepatocytesat any concentration tested.

FIGS. 20A-D show apoptosis assay data for various concentrations ofozanimod and no-drug control for primary human hepatocytes. FIG. 20Ashows the results for no-drug control. FIG. 20B shows the results fortreatment with 2.5 μM ozanimod. FIG. 20C shows the results for treatmentwith 5 μM ozanimod. FIG. 20D shows the results for treatment with 10 μMozanimod. Ozanimod did not induce apoptosis of the primary humanhepatocytes at any concentration tested.

FIG. 21 shows the cell viability effects of 5 μM SK1-I alone, 5 μMozanimod alone, and the combination of 5 μM SK1-I and 5 μM ozanimod onJurkat cells, Jurkat cells cultured with IL-2, PBMCs, and PBMCs culturedwith IL-2. The data shows, for example, that ozanimod strongly decreasesviability of Jurkat cells without IL-2 and the combination of SK1-I andozanimod even more dramatically decreases viability of Jurkat cells bothwithout and with IL-2, while the combination of agents was not nearly asdetrimental to peripheral blood mononuclear cells (PBMC) irrespective ofIL-2.

FIG. 22A shows the cell viability effects of different concentrations ofSK1-I and ozanimod, each alone and in combination, on pancreatic cellline PanC-1 cells.

FIG. 22B shows the cell viability effects of different concentrations ofSK1-I and ozanimod, each alone and in combination, on pancreatic cancercell line BxPC-3 cells.

Without limitation, the following embodiments are also provided.

Embodiments Involving Sphingosine Kinase 1 (SphK1) Inhibitors

Embodiment 1. A method for treating liver cancer in a mammalian subject,such as a human, including the step of:

-   -   administering to a mammalian subject in need of treatment for        liver cancer, an effective amount of a sphingosine kinase type I        inhibitor.

Embodiment 2. The method of embodiment 1, wherein the liver cancer ishepatic cell carcinoma (HCC).

Embodiment 3. The pharmaceutical composition of embodiment 1, whereinthe liver cancer is selected from the group consisting of fibrolamellarHCC, cholangiocarcinoma (bile duct cancer) and angiosarcoma.

Embodiment 4. The method of any one of the preceding embodiments,wherein the sphingosine kinase type I inhibitor at least substantiallydoes not inhibit sphingosine kinase type II.

Embodiment 5. The method of any one of the preceding embodiments,wherein the sphingosine kinase type I inhibitor includes a sphingosinekinase type I inhibitor disclosed in U.S. Pat. Nos. 8,372,888 and/or8,314,151, or a pharmaceutically acceptable salt thereof.

Embodiment 6. The method of any one of embodiments 1-5, wherein thesphingosine kinase type I inhibitor includes SK1-I or a pharmaceuticallyacceptable salt thereof.

Embodiment 7. The method of any one of the preceding embodiments,wherein said administration includes parenteral administration.

Embodiment 8. The method of embodiment 7, wherein said administration isvia injection, such as intravenous injection, intramuscular injection,or subcutaneous injection.

Embodiment 9. The method of any one of embodiments 1-6, wherein saidadministration includes non-parenteral administration.

Embodiment 10. The method of any one of embodiments 1-6, wherein saidadministration includes oral administration by ingestion.

Embodiment 11. The method of embodiment 10, wherein said oraladministration includes administering a dosage form including thesphingosine kinase type I inhibitor and at least one pharmaceuticallyacceptable excipient.

Embodiment 12. The method of embodiment 11, wherein the dosage form isselected from the group consisting of a tablet, a capsule, and a gelcap.

Embodiment 13. The method of any one of embodiments 1-6, wherein saidadministration includes administration via the alimentary canal.

Embodiment 14. The method of any one of the preceding embodiments,further including the step of:

-   -   co-administering to the subject an effective amount of ozanimod        or a pharmaceutically acceptable salt thereof.

Embodiment 15. A pharmaceutical composition for the treatment of a livercancer in a mammalian subject, such as a human, including:

-   -   a therapeutically effective amount of a sphingosine kinase type        I inhibitor.

Embodiment 16. The pharmaceutical composition of embodiment 15, whereinthe liver cancer is hepatic cell carcinoma (HCC).

Embodiment 17. The pharmaceutical composition of embodiment 15, whereinthe liver cancer is selected from the group consisting of FibrolamellarHCC, Cholangiocarcinoma (bile duct cancer) and Angiosarcoma.

Embodiment 18. The pharmaceutical composition of any one of thepreceding embodiments, wherein the sphingosine kinase type I inhibitorat least substantially does not inhibit sphingosine kinase type II.

Embodiment 19. The pharmaceutical composition of any one of thepreceding embodiments, wherein the sphingosine kinase type I inhibitorincludes a sphingosine kinase type I inhibitor disclosed in U.S. Pat.Nos. 8,372,888 and/or 8,314,151, or a pharmaceutically acceptable saltthereof.

Embodiment 20. The pharmaceutical composition of any one of embodiment15-18, wherein the sphingosine kinase type I inhibitor includes SK1-I ora pharmaceutically acceptable salt thereof.

Embodiment 21. The pharmaceutical composition of any one of thepreceding embodiments, wherein said composition is for parenteraladministration.

Embodiment 22. The pharmaceutical composition of embodiment 21, whereinsaid composition is for administration via injection, such asintravenous injection, intramuscular injection, or subcutaneousinjection.

Embodiment 23. The pharmaceutical composition of any one of embodiments15-20, wherein said composition is for non-parenteral administration.

Embodiment 24. The pharmaceutical composition of any one of embodiments15-20, wherein said composition is for oral administration by ingestion.

Embodiment 25. The pharmaceutical composition of any one of embodiment15-24, further including at least one pharmaceutically acceptableexcipient.

Embodiment 26. The pharmaceutical composition of any one of embodiments15-25, wherein said composition is a solid dosage form.

Embodiment 27. The pharmaceutical composition of embodiment 24, providedin a dosage form selected from the group consisting of a liquid, atablet, a capsule, and a gel cap.

Embodiment 28. The pharmaceutical composition of any one of embodiments15-20, wherein said composition is for administration via the alimentarycanal.

Embodiment 29. The pharmaceutical composition of any one of embodiments15-28, further including a therapeutically effective amount of ozanimodor a pharmaceutically acceptable salt thereof.

Embodiment 30. A method for inducing apoptosis of mammalian liver cancercells, such as hepatocellular carcinoma (HCC) cells, including the stepof:

-   -   contacting the mammalian liver cancer cells with an effective        amount of a selective sphingosine kinase type I inhibitor, such        as any of those disclosed in U.S. Pat. Nos. 8,372,888 and/or        8,314,151, such as SK1-I, or a pharmaceutically acceptable salt        thereof.

Embodiment 31. Use of a selective sphingosine kinase type I inhibitor,such as any of those disclosed in U.S. Pat. Nos. 8,372,888 and/or8,314,151, such as SK1-I, or a pharmaceutically acceptable salt thereof,for inducing apoptosis of mammalian liver cancer cells, such ashepatocellular carcinoma (HCC) cells.

Embodiments Involving Sphingosine-1-Phosphate Receptor Agonists

Embodiment 32. A method for treating a cancer or a myleoproilferativedisorder (myeloproliferative neoplasm) in a mammalian subject, such as ahuman, including the step of:

-   -   administering to a mammalian subject in need of treatment for a        cancer or myeloproliferative disorder, a therapeutically        effective amount of a sphingosine-1-phosphate receptor agonist,        such as an agonist of one or both of sphingosine-1-phosphate        receptor-1 (S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅)        such as ozanimod (RPC1063) or a pharmaceutically acceptable salt        (such as but not limited to a hydrochloride salt), ester,        prodrug, homolog, hydrate or solvate thereof, or an active        metabolite of ozanimod or a pharmaceutically acceptable salt        (such as but not limited to a hydrochloride salt), ester,        prodrug, homolog, hydrate or solvate thereof.

Embodiment 33. The method of embodiment 32, wherein themyeloproliferative disorders is selected from the group consisting of:Chronic myelogenous leukemia (e.g, BCR-ABL1-positive); Chronicneutrophilic leukemia; Polycythemia vera; Primary myelofibrosis;Essential thrombocythemia; Chronic eosinophilic leukemia (not otherwisespecified); and Mastocytosis.

Embodiment 34. The method of embodiment 32, wherein said cancer is ahematological malignancy.

Embodiment 35. The method of embodiment 34, wherein said hematologicalmalignancy is selected from the group consisting of: leukemias,lymphomas and myelomas.

Embodiment 36. The method of embodiment 35, wherein said hematologicalmalignancy is selected from the group consisting of: Acute lymphoblasticleukemia (ALL); Acute myelogenous leukemia (AML); Chronic lymphocyticleukemia (CLL); Chronic myelogenous leukemia (CML); Acute monocyticleukemia (AMoL); Hodgkin's lymphomas (e.g., any of main four subtypes);and Non-Hodgkin's lymphomas (any subtype).

Embodiment 37. The method of embodiment of embodiment 32, wherein saidcancer is a solid organ cancer.

Embodiment 38. The method of embodiment 32, wherein the solid organcancer is selected from the group consisting of: Adipose tissue cancerssuch as Liposarcoma, Myxoid liposarcoma adipose; Bladder cancer; Bonecancers such as Chondroblastoma, Chordoma, Ewings sarcoma, Osteosarcoma,Spindle cell tumor; Brain tumors such as Ganglioneuroblastoma,Ganglioneuroma, Glioblastoma, Malignant peripheral nerve sheath tumor,Neuroblastoma, Neurofibroma, Schwannoma brain; Connective tissue cancerssuch as Chondromyxoid fibroma, Chondrosarcoma, Dedifferentiatedchondrosarcoma, Fibromatosis, Monophasic synovial sarcoma; Esophagealadenocarcinoma; Oral squamous cell carcinoma; Kidney cancers such asKidney carcinoma, Renal cell carcinoma; Liver cancers such asHepatocellular carcinoma (HCC), Fibrolamellar HCC, Cholangiocarcinoma(bile duct cancer) and Angiosarcoma; Lung cancer such as NSCLC, SCLC;Uterine tumors; Head and Neck cancers such as head and neck squamouscell carcinoma; Ovarian tumors; Prostate cancer; Muscle tissue cancerssuch as Acute quadriplegic myopathy; Skin cancers such as Melanoma,Sarcoma, Kaposi sarcoma; Alveolar rhabdomyo sarcoma, Embryonal rhabdomyosarcoma, Leiomyosarcoma; Germ cell tumors such as of the testes,testicular cancer; Thyroid cancer such as Thyroid adenocarcinoma; andPancreatic cancer.

Embodiment 39. The method of embodiment 33, wherein the solid organcancer is liver cancer.

Embodiment 40. The method of embodiment 39, wherein the liver cancer ishepatic cell carcinoma (HCC).

Embodiment 41. The method of any one of embodiments 32-40, wherein thesphingosine-1-phosphate receptor agonist at least substantially does notagonize sphingosine-1-phosphate receptors other than types-1 and -5.

Embodiment 42. The method of any one of embodiments 32-41, wherein thesphingosine-1-phosphate receptor agonist includes asphingosine-1-phosphate receptor agonist disclosed in any of U.S. Pub.Nos. 20110172202, 20130231326, and 20150299149 or a pharmaceuticallyacceptable salt (such as but not limited to a hydrochloride salt),ester, prodrug, homolog, hydrate or solvate thereof.

Embodiment 43. The method of any one of embodiments 32-42, wherein thesphingosine-1-phosphate receptor agonist includes ozanimod or apharmaceutically acceptable salt (such as but not limited to ahydrochloride salt), ester, prodrug, homolog, hydrate or solvatethereof.

Embodiment 44. The method of any one of embodiments 32-43, wherein saidadministration includes parenteral administration.

Embodiment 45. The method of embodiment 44, wherein said administrationis via injection, such as intravenous injection, intramuscularinjection, or subcutaneous injection.

Embodiment 46. The method of any one of embodiments 32-43, wherein saidadministration includes non-parenteral administration.

Embodiment 47. The method of any one of embodiments 32-43, wherein saidadministration includes oral administration by ingestion.

Embodiment 48. The method of embodiment 47, wherein said oraladministration includes administering a dosage form including thesphingosine-1-phosphate receptor agonist and at least onepharmaceutically acceptable excipient.

Embodiment 49. The method of embodiment 48, wherein the dosage form isselected from the group consisting of a liquid, a tablet, a capsule, anda gel cap.

Embodiment 50. The method of any one of embodiments 32-43, wherein saidadministration includes administration via the alimentary canal.

Embodiment 51. The method of any one of embodiments 32-50, furtherincluding the step of:

-   -   co-administering to the subject an effective amount of a        sphingosine kinase type I inhibitor, such as SK1-I, or a        pharmaceutically acceptable salt thereof.

Embodiment 52. The method of any one of embodiments 32-51, furtherincluding the step of:

-   -   co-administering to the subject a therapeutically effective        amount of a cellular ceramide generation promoter, such as        6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic        acid]cyclosporin D (Valspodor; PSC833) or a pharmaceutically        acceptable salt thereof.

Embodiment 53. The method of any one of embodiments 32-52, furtherincluding the step of:

-   -   coadministering to the subject a therapeutically effective        amount of ceramide.

Embodiment 54. A pharmaceutical composition for the treatment of acancer or a myeloproliferative disorder (myeloproliferative neoplasm) ina mammalian subject, such as a human, including:

-   -   a therapeutically effective amount of a sphingosine-1-phosphate        receptor agonist, such as an agonist of one or both of        sphingosine-1-phosphate receptor-1 (S1P₁) and        sphingosine-1-phosphate receptor-5 (S1P₅) such as ozanimod        (RPC1063) or a pharmaceutically acceptable salt (such as but not        limited to a hydrochloride salt), ester, prodrug, homolog,        hydrate or solvate thereof.

Embodiment 55. The pharmaceutical composition of embodiment 54, whereinthe myeloproliferative disorders is selected from the group consistingof: Chronic myelogenous leukemia (BCR-ABL1-positive); Chronicneutrophilic leukemia; Polycythemia vera; Primary myelofibrosis;Essential thrombocythemia; Chronic eosinophilic leukemia (not otherwisespecified); and Mastocytosis.

Embodiment 56. The pharmaceutical composition of embodiment 54, whereinsaid cancer is a hematological malignancy.

Embodiment 57. The pharmaceutical composition of embodiment 56, whereinsaid hematological malignancy is selected from the group consisting of:leukemias, lymphomas and myelomas.

Embodiment 58. The pharmaceutical composition of embodiment 57, whereinsaid hematological malignancy is selected from the group consisting of:Acute lymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML);Chronic lymphocytic leukemia (CLL); Chronic myelogenous leukemia (CML);Acute monocytic leukemia (AMoL); Hodgkin's lymphomas (e.g., any of mainfour subtypes); and Non-Hodgkin's lymphomas (any subtype).

Embodiment 59. The pharmaceutical composition of embodiment ofembodiment 54, wherein said cancer is a solid organ cancer.

Embodiment 60. The pharmaceutical composition of embodiment 59, whereinthe solid organ cancer is selected from the group consisting of: Adiposetissue cancers such as Liposarcoma, Myxoid liposarcoma adipose; Bladdercancer; Bone cancers such as Chondroblastoma, Chordoma, Ewings sarcoma,Osteosarcoma, Spindle cell tumor; Brain tumors such asGanglioneuroblastoma, Ganglioneuroma, Malignant peripheral nerve sheathtumor, Neuroblastoma, Neurofibroma, Schwannoma brain; Connective tissuecancers such as Chondromyxoid fibroma, Chondrosarcoma, Dedifferentiatedchondrosarcoma, Fibromatosis, Monophasic synovial sarcoma; Esophagealadenocarcinoma; Oral squamous cell carcinoma; Kidney cancers such asKidney carcinoma, Renal cell carcinoma; Liver cancers such asHepatocellular carcinoma (HCC), Fibrolamellar HCC, Cholangiocarcinoma(bile duct cancer) and Angiosarcoma; Lung cancer such as NSCLC, SCLC;Uterine tumors; Head and Neck cancers such as head and neck squamouscell carcinoma; Ovarian tumors; Prostate cancer; Muscle tissue cancerssuch as Acute quadriplegic myopathy; Skin cancers such as Melanoma,Sarcoma, Kaposi sarcoma; Alveolar rhabdomyo sarcoma, Embryonal rhabdomyosarcoma, Leiomyosarcoma; Germ cell tumors such as of the testes,testicular cancer; Thyroid cancer such as Thyroid adenocarcinoma; andPancreatic cancer.

Embodiment 61. The pharmaceutical composition of embodiment 59, whereinthe solid organ cancer is a liver cancer.

Embodiment 62. The pharmaceutical composition of embodiment 61, whereinthe liver cancer is selected from the group consisting of hepatic cellcarcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma (bile ductcancer) and angiosarcoma.

Embodiment 63. The pharmaceutical composition of any one of embodiments54-62, wherein the sphingosine-1-phosphate receptor agonist at leastsubstantially does not agonize sphingosine-1-phosphate receptors otherthan types-1 and -5.

Embodiment 65. The pharmaceutical composition of any one of embodiments54-64, wherein the sphingosine-1-phosphate receptor agonist includes asphingosine-1-phosphate receptor agonist disclosed in any of U.S. PubNos. 20110172202, 20130231326, and 20150299149.

Embodiment 66. The pharmaceutical composition of any one of embodiments54-64, wherein the sphingosine-1-phosphate receptor agonist includesozanimod or a pharmaceutically acceptable salt thereof.

Embodiment 67. The pharmaceutical composition of any one of embodiments54-66, wherein said composition is for parenteral administration.

Embodiment 68. The pharmaceutical composition of embodiment 67, whereinsaid composition is for administration via injection, such asintravenous injection, intramuscular injection, or subcutaneousinjection.

Embodiment 69. The pharmaceutical composition of any one of embodiments54-66, wherein said composition is for non-parenteral administration.

Embodiment 70. The pharmaceutical composition of any one of embodiments54-66, wherein said composition is for oral administration by ingestion.

Embodiment 71. The pharmaceutical composition of any one of embodiments54-70, further including at least one pharmaceutically acceptableexcipient.

Embodiment 72. The pharmaceutical composition of any one of embodiments54-71, wherein said composition is a solid dosage form.

Embodiment 73. The pharmaceutical composition of embodiment 72, providedin a dosage form selected from the group consisting of a tablet, acapsule, and a gel cap.

Embodiment 74. The pharmaceutical composition of any one of embodiments54-66, wherein said composition is for administration via the alimentarycanal.

Embodiment 75. The pharmaceutical composition of any one of embodiments54-74, further including a therapeutically effective amount of asphingosine kinase type I inhibitor, such as one disclosed in U.S. Pat.Nos. 8,372,888 and/or 8,314,151, such as SK1-I, or a pharmaceuticallyacceptable salt thereof.

Embodiment 76. The pharmaceutical composition of any one of embodiments54-74, for use in combination with a therapeutically effective amount ofa sphingosine kinase type I inhibitor, such as one disclosed in U.S.Pat. Nos. 8,372,888 and/or 8,314,151, such as SK1-I, or apharmaceutically acceptable salt thereof.

Embodiment 77. The pharmaceutical composition of any one of embodiments54-76, further including a therapeutically effective amount of acellular ceramide generation promoter, such as6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporinD (Valspodor; PSC833) or a pharmaceutically acceptable salt thereof.

Embodiment 78. The pharmaceutical composition of any one of embodiments54-76, for use in combination with a therapeutically effective amount ofa cellular ceramide generation promoter,6-[(2S,4R,6E)-4-Methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporinD (Valspodor; PSC833) or a pharmaceutically acceptable salt thereof.

Embodiment 79. The pharmaceutical composition of any one of embodiments54-78 for use in combination with a therapeutically effective amount ofceramide.

Embodiment 80. A method for inducing apoptosis of mammalian cancercells, such as liver cancer cells, such as hepatocellular carcinoma(HCC) cells, including the step of:

-   -   contacting the mammalian cancer cells with an effective amount        of a sphingosine-1-phosphate receptor agonist, such as an        agonist of one or both of sphingosine-1-phosphate receptor-1        (S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅) such as any        of those disclosed in U.S. Pub Nos. 20110172202, 20130231326,        and 20150299149, such as ozanimod (RPC1063) or a        pharmaceutically acceptable salt (such as but not limited to a        hydrochloride salt), ester, prodrug, homolog, hydrate or solvate        thereof, or an active metabolite of ozanimod or a        pharmaceutically acceptable salt (such as but not limited to a        hydrochloride salt), ester, prodrug, homolog, hydrate or solvate        thereof.

Embodiment 81. Use of a sphingosine-1-phosphate receptor agonist, suchas an agonist of one or both of sphingosine-1-phosphate receptor-1(S1P₁) and sphingosine-1-phosphate receptor-5 (S1P₅) such as any ofthose disclosed in U.S. Pub Nos. 20110172202, and 20130231326, and20150299149, such as ozanimod (RPC1063), or a pharmaceuticallyacceptable salt (such as but not limited to a hydrochloride salt),ester, prodrug, homolog, hydrate or solvate thereof, for inducingapoptosis of mammalian cancer cells, such as liver cancer cells, suchas, hepatocellular carcinoma (HCC) cells, or an active metabolite ofozanimod or a pharmaceutically acceptable salt (such as but not limitedto a hydrochloride salt), ester, prodrug, homolog, hydrate or solvatethereof.

Embodiment 82. Any of embodiments 32-42, 44-65 and 67-81 wherein thesphingosine-1-phosphate receptor agonist is a compound having thestructure:

wherein,

-   -   X is —NR′R″ or —OR′″;    -   Y is —CN, —Cl, or —CF₃;    -   R′ is H, C₁₋₄ alkyl, n-hydroxy C₁₋₄ alkyl, —SO₂—R¹, or —CO—R¹;    -   R″ is H, —SO₂—R³, C₁₋₄ alkyl optionally substituted with 1 or        more R², or a ring moiety optionally substituted with R⁴ wherein        such ring moiety is piperidinyl, cyclohexyl, morpholinyl,        pyrrolidinyl, imidazolyl, or phenyl;    -   R′″ is H, C₁₋₄ alkyl, or —CO—R′;        -   or alternatively, R′ and R″ taken together with the nitrogen            atom to which they are bound form a 4-, 5-, or 6-membered            saturated heterocyclic ring containing 0 or 1 additional            heteroatoms where such additional heteroatom is O or N            wherein such heterocycle is optionally singly or multiply            substituted with substituents independently selected from            —OH, oxo, —NH₂, n-hydroxy-C₁₋₄ alkyl, —COOH,            —(CH₂)_(m)—COOH, —(CH₂)_(m)—COOR¹, —N(R¹R¹), and            —(CH₂)_(m)—CO—N(R⁵R⁵);    -   each R¹ is independently C₁₋₄ alkyl or H;    -   each R² is independently H, halo, OH, oxo, ═NH, NH₂, —COOH, F,        —NHR¹, —N(R⁵R⁵), —SO₂—R¹, —SO₂—N(R⁵R⁵), —N(R¹)—SO₂—R¹, —COOR¹,        —OCO—R¹, —CO—N(R⁵R⁵), —N(R¹)—COR¹, C₁₋₃ alkyl, C₁₋₃ alkoxy, and        a ring moiety optionally substituted with R⁴ wherein such ring        moiety is piperazinyl, piperidinyl, morpholinyl, pyrrolidinyl,        pyrazolyl, imidazolyl, benzimidazolyl, azetidinyl, cyclobutinyl,        or phenyl;    -   each R³ is independently R², C₁₋₄ alkyl, C₃₋₆ cycloalkyl, or        C₁₋₄ alkyl optionally substituted with 1 or more R²;    -   each R⁴ is independently halo, OH, —NH₂, —NHR¹, —N(R¹R¹), —COOH,        —COOR¹, —NHCO—R¹;    -   each R⁵ is independently C₁₋₄ alkyl or H, or alternatively two        R⁵ taken together with the nitrogen atom to which they are bound        can form a 4-, 5-, or 6-membered saturated heterocyclic ring        containing 0 or 1 additional heteroatoms where such additional        heteroatom is O or N wherein such heterocycle is optionally        substituted with —OH, NH₂, —N(R¹R¹), n-hydroxy C₁₋₄ alkyl,        —(CH₂)_(m)—COOH, or —(CH₂)_(m)—COOR¹; and each m is        independently 0, 1, 2, or 3, or        a pharmaceutically acceptable salt (such as but not limited to a        hydrochloride salt), ester, prodrug, homolog, hydrate or solvate        thereof.

Embodiment 83. Any of embodiments 32-42, 44-65 and 67-82 wherein thesphingosine-1-phosphate receptor agonist is a compound having thestructure:

or a pharmaceutically acceptable salt (such as but not limited to ahydrochloride salt), ester, prodrug, homolog, hydrate or solvatethereof. The compounds shown are active metabolites of ozanimod. X mayhave an (R) or (S) configuration where not specified.

Embodiment 84. Any of embodiments 32-42, 44-65 and 67-81 wherein thesphingosine-1-phosphate receptor agonist is the ozanimod metaboliteCC-112273 or a pharmaceutically acceptable salt (such as but not limitedto a hydrochloride salt), ester, prodrug, homolog, hydrate or solvatethereof.

Embodiment 85. A method for treating a cancer or myeloproliferativedisorder, such as any of those described herein, in a mammal such as ahuman being, in need of treatment thereof, including administering tothe mammal a therapeutically effective amount of a compound having thestructure:

wherein,

-   -   X is —NR′R″ or —OR′″;    -   Y is —CN, —Cl, or —CF₃;    -   R′ is H, C₁₋₄ alkyl, n-hydroxy C₁₋₄ alkyl, —SO₂—R¹, or —CO—R¹;    -   R″ is H, —SO₂—R³, C₁₋₄ alkyl optionally substituted with 1 or        more R², or a ring moiety optionally substituted with R⁴ wherein        such ring moiety is piperidinyl, cyclohexyl, morpholinyl,        pyrrolidinyl, imidazolyl, or phenyl;    -   R′″ is H, C₁₋₄ alkyl, or —CO—R¹;        -   or alternatively, R′ and R″ taken together with the nitrogen            atom to which they are bound form a 4-, 5-, or 6-membered            saturated heterocyclic ring containing 0 or 1 additional            heteroatoms where such additional heteroatom is O or N            wherein such heterocycle is optionally singly or multiply            substituted with substituents independently selected from            —OH, oxo, —NH₂, n-hydroxy-C₁₋₄ alkyl, —COOH,            —(CH₂)_(m)—COOH, —(CH₂)_(m)—COOR¹, —N(R¹R¹), and            —(CH₂)_(m)—CO—N(R⁵R⁵);    -   each R¹ is independently C₁₋₄ alkyl or H;    -   each R² is independently H, halo, OH, oxo, ═NH, NH₂, —COOH, F,        —NHR¹, —N(R⁵R⁵), —SO₂—R¹, —SO₂—N(R⁵R⁵), —N(R¹)—SO₂—R¹, —COOR¹,        —OCO—R¹, —CO—N(R⁵R⁵), —N(R¹)—COR¹, C₁₋₃ alkyl, C₁₋₃ alkoxy, and        a ring moiety optionally substituted with R⁴ wherein such ring        moiety is piperazinyl, piperidinyl, morpholinyl, pyrrolidinyl,        pyrazolyl, imidazolyl, benzimidazolyl, azetidinyl, cyclobutinyl,        or phenyl;    -   each R³ is independently R², C₁₋₄ alkyl, C₃₋₆ cycloalkyl, or        C₁₋₄ alkyl optionally substituted with 1 or more R²;    -   each R⁴ is independently halo, OH, —NH₂, —NHR¹, —N(R¹R¹), —COOH,        —COOR¹, —NHCO—R¹;    -   each R⁵ is independently C₁₋₄ alkyl or H, or alternatively two        R⁵ taken together with the nitrogen atom to which they are bound        can form a 4-, 5-, or 6-membered saturated heterocyclic ring        containing 0 or 1 additional heteroatoms where such additional        heteroatom is O or N wherein such heterocycle is optionally        substituted with —OH, NH₂, —N(R¹R¹), n-hydroxy C₁₋₄ alkyl,        —(CH₂)_(m)—COOH, or —(CH₂)_(m)—COOR¹; and        -   each m is independently 0, 1, 2, or 3, or            a pharmaceutically acceptable salt (such as but not limited            to a hydrochloride salt), ester, prodrug, homolog, hydrate            or solvate thereof.

Embodiment 86. Use of a compound or pharmaceutically acceptable salt(such as but not limited to a hydrochloride salt), ester, prodrug,homolog, hydrate or solvate thereof as set forth in embodiment 85 in thetreatment of a cancer or myeloproliferative disorder, such as any ofthose described herein, in a mammal such as a human being.

Embodiment 87. A pharmaceutical composition for the treatment of acancer or myeloproliferative disorder, such as any of those describedherein, in a mammal such as a human being, the composition including atherapeutically effective amount of a compound or pharmaceuticallyacceptable salt (such as but not limited to a hydrochloride salt),ester, prodrug, homolog, hydrate or solvate thereof as set forth inembodiment 85, and optionally further including at least onepharmaceutically acceptable excipient.

Embodiment 88. Any of embodiments 85-87 wherein the compound has thestructure:

X may have an (R) or (S) configuration where not specified.

Embodiment 89. A method for treating a cancer or myeloproliferativedisorder, such as any of those described herein, in a mammal such as ahuman being, in need of treatment thereof, including administering tothe mammal a therapeutically effective amount of the ozanimod metaboliteCC-112273 or a pharmaceutically acceptable salt (such as but not limitedto a hydrochloride salt), ester, prodrug, homolog, hydrate or solvatethereof.

Embodiment 90. Use of the ozanimod metabolite CC-112273 or apharmaceutically acceptable salt (such as but not limited to ahydrochloride salt), ester, prodrug, homolog, hydrate or solvate thereofin the treatment of a cancer or myeloproliferative disorder, such as anyof those described herein, in a mammal such as a human being.

Embodiment 91. A pharmaceutical composition for the treatment of acancer or myeloproliferative disorder, such as any of those describedherein, in a mammal such as a human being, the composition including atherapeutically effective amount of the ozanimod metabolite CC-112273 ora pharmaceutically acceptable salt (such as but not limited to ahydrochloride salt), ester, prodrug, homolog, hydrate or solvatethereof.

Embodiment 92. Any of embodiments of 82 and 85-85, wherein X is —NH₂,

For embodiments involving ceramide, the ceramide may, for example, beformulated/co-formulated in an acid stable lipid vesicle/particlecomposition as disclosed in U.S. Pub. No. 20140271824, which is herebyincorporated by reference in its entirety, andadministered/co-administered, for example by injection, such asintravenous injection, or orally.

Any of the method of treatment/use embodiments set forth herein mayfurther include the step of: co-administering one or more immunecheckpoint inhibitors, which may be monoclonal antibodies (mABs). Theimmune checkpoint inhibitor may be selected from the group consisting ofthe following: PD-1 inhibitors such as mAbs Pembrolizumab (Keytruda®)and Nivolumab (Opdivo®); PD-L1 inhibitors such as mAbs Atezolizumab(Tecentriq®), Avelumab (Bavencio®), and Durvalumab (Imfinzi®); andCTLA-4 inhibitors such as mAb Ipilimumab (Yervoy®); and V-domain IgSuppressor of T Cell Activation (VISTA) inhibitors such as mAbJNJ-61610588 (ImmuNext Inc.). Similarly, any of the pharmaceuticalcomposition embodiments of the invention may be for use in combinationwith one or more immune checkpoint inhibitors, such as those disclosedherein.

Still another embodiment of the invention provides a method for treatinga cancer or a myleoproliferative disorder (myeloproliferative neoplasm),such as any of those disclosed herein, for example, a liver cancer, in amammalian subject, such as a human, including the step of:

-   -   co-administering to a mammalian subject in need of treatment for        a cancer or myeloproliferative disorder, a therapeutically        effective amount of:        -   (a) a sphingosine kinase type I inhibitor, such as one            disclosed in U.S. Pat. Nos. 8,372,888 and/or 8,314,151, such            as SK1-I, or a pharmaceutically acceptable salt thereof; and        -   (b) one or more immune checkpoint inhibitors, which may be            monoclonal antibodies, such as one or more selected from the            group consisting of: PD-1 inhibitors such as mAbs            Pembrolizumab (Keytruda®) and Nivolumab (Opdivo®); PD-L1            inhibitors such as mAbs Atezolizumab (Tecentriq®), Avelumab            (Bavencio®), and Durvalumab (Imfinzi®); and CTLA-4            inhibitors such as mAb Ipilimumab (Yervoy®); and V-domain Ig            Suppressor of T Cell Activation (VISTA) inhibitors such as            mAb JNJ-61610588 (ImmuNext Inc.).

Immune checkpoint inhibitors may, for example, be administered byinjection in the dosages described herein and/or at the currentlyapproved dosages for said inhibitors.

The amount of compound that is effective for the treatment or preventionof a condition, alone or in combination with other compounds, may bedetermined by standard techniques. In addition, in vitro and/or in vivoassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed will also depend on, e.g., theroute of administration and the seriousness of the condition, and can bedecided according to the judgment of a practitioner and/or eachpatient's circumstances. In other examples thereof, variations willnecessarily occur depending upon the weight and physical condition(e.g., hepatic and renal function) of the patient being treated, theaffliction to be treated, the severity of the symptoms, the frequency ofthe dosage interval, the presence of any deleterious side-effects, andthe particular compound utilized, among other things.

Administration may be as a single dose or as a divided dose. In oneembodiment, an effective dosage is administered once per month until thecondition is abated. In another embodiment, the effective dosage isadministered once per week, or twice per week or three times per weekuntil the condition is abated. An effective dosage may, for example, beadministered at least once daily or at least or at least once everytwo-days, or at least once every three days, four days, five days, sixdays or seven days. In another embodiment, an effective dosage amount isadministered about every 24 h until the condition is abated. In anotherembodiment, an effective dosage amount is administered about every 12 huntil the condition is abated. In another embodiment, an effectivedosage amount is administered about every 8 h until the condition isabated. In another embodiment, an effective dosage amount isadministered about every 6 h until the condition is abated. In anotherembodiment, an effective dosage amount is administered about every 4 huntil the condition is abated.

The therapeutically effective doses/amounts of the pharmaceuticalcompounds disclosed herein may be expressed in terms of the amount ofthe compound(s) or pharmaceutically acceptable salts thereofadministered per unit body weight of the subject per day of treatment,or the total amount administered per day of treatment. A daily dose may,for example, be at least 0.005 mg/kg of body weight, at least 0.01 mg/kgof body weight, at least 0.025 mg/kg of body weight, at least 0.05 mg/kgof body weight, at least 0.1 mg/kg of body weight, at least 0.2 mg/kg ofbody weight, at least 0.3 mg/kg of body weight, at least 0.4 mg/kg ofbody weight, at least 0.5 mg/kg of body weight, at least 0.6 mg/kg ofbody weight, at least 0.7 mg/kg of body weight, at least 0.8 mg/kg ofbody weight, at least 0.9 mg/kg of body weight, at least 1 mg/kg of bodyweight, at least 1.5 mg/kg of body weight, at least 2 mg/kg of bodyweight, at least 2.5 mg/kg of body weight, at least 3 mg/kg of bodyweight, at least 3.5 mg/kg of body weight, at least 4 mg/kg of bodyweight, at least 4.5 mg/kg of body weight, at least 5 mg/kg of bodyweight, or at one of said doses. A total daily dose may, for example, bein the range of 0.005 mg/kg to 5 mg/kg or any subrange or value therein,such as 0.025 to 5 mg/kg body weight, such as 0.05 to 5 mg/kg bodyweight. A total daily dose may, for example be in the range of 0.1 mg to1,000 mg total or any subrange or value therein, such as 0.1 mg to 1,000mg, such as 0.1 mg to 100 mg, such as 0.1 mg to 50 mg, such as 0.5 mg to50 mg, such as 1.0 mg to 50 mg, such as 5 mg to 50 mg, or 0.1 mg to 10mg, such as 0.5 mg to 10 mg. For SK1-I and related SphK1 inhibitorsdisclosed U.S. Pat. Nos. 8,372,888 and 8,314,151, and pharmaceuticallyacceptable salts thereof, a daily dose for human subjects may, forexample, also be in the range of 0.5 mg/kg to 5 mg/kg or any subrange orvalue therein, such as 1 mg/kg to 4 mg/kg, such as 1 mg/kg to 3 mg/kg,or, for example, a total daily dose of 5 mg to 50 mg or any subrange orvalue therein, such as 10 mg to 40 mg, such as 20 mg to 40 mg. Forozanimod, its active metabolites and related sphingosine-1-phosphatereceptor agonists disclosed in U.S. Pub Nos. 20110172202, 20130231326,and 20150299149, and pharmaceutically acceptable salts thereof, a dailydose for human subjects may, for example, also be in the range of 1 mgto 50 mg or any subrange or value therein, or 0.1 mg to 10 mg or anysubrange or value therein, such as 0.1 mg to 5 mg, such as 0.5 to 5 mg,such as 0.5 mg to 2.5 mg, such as 0.5 mg to 1.5 mg. A pharmaceuticalcomposition according to the invention may, for example, include a dailydose amount of the compound as set forth herein.

The duration of treatment by administration of a therapeutic compound orcombination according to the invention may continue for a plurality ofdays, such as for at least one week, at least two weeks, at least threeweeks, at least four weeks, at least two months, at least three months,at least four months, at least five months, at least six months, atleast seven months, at least eight months, at least nine months, atleast 10 months, at least 11 months, at least 12 months, at least 1½years, at least 2 years, at least three years, at least four years, ormay continue indefinitely.

The terms co-administration and co-administering mean that each of thethings being co-administered is administered to a subject in suchtemporal proximity that each (or its active metabolite(s)) is present inactive form in the subject for an at least partially overlapping periodof time. Accordingly, co-administration may include, simultaneousadministration, such as when the things being administered are part ofthe same pharmaceutical composition, or sequential administration of thethings being co-administered, for example, within the same day of eachother, within 12 hours of each other, within 6 hours of each other,within 3 hours of each other, within 1 hours of each other, or within 15minutes of each other. The things being administered may be administeredby the same route, such as by oral ingestion or injection, or bydifferent routes.

Pharmaceutically acceptable salts and the selection and preparationthereof are well known in the art. Such salts include but are notlimited to hydrochloride, citrate, glycolate, fumarate, malate,tartrate, mesylate, esylate, cinnamate, isethionate, sulfate, phosphate,diphosphate, nitrate, hydrobromide, hydroiodide, succinate, formate,acetate, dichloroacetate, lactate, p-toluenesulfonate, pamitate,pidolate, pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamidobenzoate, glutamate, aspartate, glycolate, adipate, alginate, ascorbate,besylate, camphorate, camphorsulfonate, camsylate, caprate, caproate,cyclamate, laurylsulfate, edisylate, gentisate, galactarate, gluceptate,gluconate, glucuronate, oxoglutarate, hippurate, lactobionate, malonate,maleate, mandalate, napsylate, napadisylate, oxalate, oleate, sebacate,stearate, succinate, thiocyanate, undecylenate, and xinafoate.

It should be noted that the indefinite articles “a” and “an” and thedefinite article “the” are used in the present application to mean oneor more unless the context clearly dictates otherwise. Further, the term“or” is used in the present application to mean the disjunctive “or” orthe conjunctive “and.” It should also be understood that wherever in thepresent application the term comprising or including (or a term ofsimilar scope) is recited in connection with the description of anyembodiment or part thereof, a corresponding embodiment or part thereofreciting instead the term consisting essentially of or the termconsisting of (or a term of similar scope) is also disclosed. It shouldalso be understood that wherever a chemical structure or chemical groupdisclosed herein has one or more stereoisomers or stereoisomeric forms,corresponding embodiments directed to each of the stereoisomers orstereoisomeric forms individually or to any combination of theparticular stereoisomers or stereoisomeric forms are also intended to bedisclosed.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s). Moreover,features described in connection with one embodiment of the inventionmay be used in conjunction with other embodiments, even if notexplicitly exemplified in combination within.

What is claimed is:
 1. A method for treating pancreatic cancer in amammalian subject, comprising the step of: administering to a mammaliansubject in need of treatment for pancreatic cancer a therapeuticallyeffective amount of ozanimod or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the mammalian subject is ahuman.
 3. The method of claim 1, wherein the administering stepcomprises administering the hydrochloride salt of ozanimod to themammalian subject.
 4. The method of claim 2, wherein the administeringstep comprises administering the hydrochloride salt of ozanimod to themammalian subject.